Aortic valve stenosis (AVS) is a progressive fibrotic disease that is caused by thickening and stiffening of valve leaflets. At the cellular level, quiescent valve interstitial cells (qVICs) activate to myofibroblasts (aVICs) that persist within the valve tissue. Given the persistence of myofibroblasts in AVS, epigenetic mechanisms have been implicated. Here, we studied changes that occur in VICs during myofibroblast activation by using a hydrogel matrix to recapitulate different stiffnesses in the valve leaflet during fibrosis. We first compared the chromatin landscape of qVICs cultured on soft hydrogels and aVICs cultured on stiff hydrogels, representing the native and diseased phenotypes respectively. Using assay for transposase-accessible chromatin sequencing (ATAC-Seq), we found that open chromatin regions in aVICs were enriched for transcription factor binding motifs associated with mechanosensing pathways compared to qVICs. Next, we used RNA-Seq to show that the open chromatin regions in aVICs correlated with pro-fibrotic gene expression, as aVICs expressed higher levels of contractile fiber genes, including myofibroblast markers such as alpha smooth muscle actin (αSMA), compared to qVICs. In contrast, chromatin remodeling genes were downregulated in aVICs compared to qVICs, indicating qVICs may be protected from myofibroblast activation through epigenetic mechanisms. Small molecule inhibition of one of these remodelers, CREB Binding Protein (CREBBP), prevented qVICs from activating to aVICs. Notably, CREBBP is more abundant in valves from healthy patients compared to fibrotic valves. Our findings reveal the role of mechanical regulation in chromatin remodeling during VIC activation and quiescence and highlight one potential therapeutic target for treating AVS.

中文翻译：

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细胞外基质硬度通过表观遗传重塑控制心脏瓣膜肌成纤维细胞活化

主动脉瓣狭窄（AVS）是一种进行性纤维化疾病，由瓣叶增厚和硬化引起。在细胞水平上，静止的瓣膜间质细胞（qVIC）激活为持续存在于瓣膜组织内的肌成纤维细胞（aVIC）。鉴于 AVS 中肌成纤维细胞的持续存在，表观遗传机制已被证实。在这里，我们通过使用水凝胶基质来重现纤维化过程中瓣膜小叶的不同硬度，研究了肌成纤维细胞激活过程中 VIC 发生的变化。我们首先比较了在软水凝胶上培养的 qVIC 和在硬水凝胶上培养的 aVIC 的染色质景观，分别代表天然表型和患病表型。通过转座酶可及染色质测序 (ATAC-Seq) 分析，我们发现与 qVIC 相比，aVIC 中的开放染色质区域富含与机械传感途径相关的转录因子结合基序。接下来，我们使用 RNA-Seq 证明 aVIC 中的开放染色质区域与促纤维化基因表达相关，因为与 qVIC 相比，aVIC 表达更高水平的收缩纤维基因，包括肌成纤维细胞标记物，如 α 平滑肌肌动蛋白 (αSMA) 。相比之下，与 qVIC 相比，aVIC 中的染色质重塑基因下调，表明 qVIC 可能通过表观遗传机制免受肌成纤维细胞激活的影响。对其中一种重塑剂 CREB ​​结合蛋白 (CREBBP) 进行小分子抑制，可阻止 qVIC 激活为 aVIC。值得注意的是，与纤维化瓣膜相比，健康患者瓣膜中的 CREBBP 含量更高。我们的研究结果揭示了 VIC 激活和静止期间染色质重塑中机械调节的作用，并强调了治疗 AVS 的一个潜在治疗靶点。